Nutrient limitation is one of the most common stresses that bacteria encounter during their "life cycle" in nature and, to a lesser extent, in host organisms. Salmonella, in particular, must endure extensive periods of nutrient starvation in natural aquatic and terrestrial environments while retaining its pathogenic potential. The goal of our research is to elucidate how Salmonella typhimurium responds to and survives these periods of nutrient deprivation. Additionally, we are interested in determining how nutrient deprivation impacts on the virulence of this organism in terms of invasion, intracellular survival and multiplication, and ability to cause systemic disease. Recent findings have provided circumstantial evidence that nutrient starvation (specifically phosphate (P)-, carbon (C)-, or nitrogen (N)-starvation) may be important in virulence of this, and other, pathogens. We have identified three loci required for bacterial survival during prolonged nutrient deprivation. These 3 loci, and an additional locus, exhibit increased expression during P-, C-, and/or N-starvation, as determined by Mud-directed lac gene fusions. These 4 loci display a complex interactive network of regulation that involves repression by the CRP protein, in both a cAMP-dependent and -independent manner. Genetic and physiologic data indicates that the induction of these 4 loci during P-, C-, or N-starvation occurs through separate pathways possessing components that are both shared and unique to each condition. The C- and N-starvation-induction of these loci is relA-dependent, while the P-starvation-induction is not. Both C- and N-starvation result in ppGpp accumulation. One should, therefore, expect that if ppGpp is required for induction during C- and N-starvation that a gene induced by C-starvation should also be induced by N-starvation, and vice versa. Amazingly, this was not always the case suggesting a model whereby ppGpp acts through separate pathways to elicit C- and N-starvation-induction. The research outlined in this proposal is designed to clarify the most intriguing aspects of the complex regulation of these starvation-inducible (sti) loci; (1) the P-starvation-specific regulatory pathway, (2) the separate relA-dependent regulatory pathways for C- and N-starvation, and (3) the cAMP-independent CRP-dependent repression of the stiB locus. This will be accomplished by identifying mutations which effect the regulation of these loci in different genetic backgrounds and during different growth conditions. These regulatory mutations will also be tested for their effect on the starvation-survival of this organism. In addition, we will begin the molecular characterization of these loci by cloning the 5'-regulatory regions of these loci using the methodology of B. L. Wanner that is designed for this purpose; or by identifying, purifying, and cloning lac containing chromosomal restriction fragments from sti-lac fusion strains, selecting for beta-galactosidase activity or the presence of lac DNA sequences, directly. Additionally, collaborative studies with Dr. B. Finlay will be performed to determine if these, and other, Mud-lac gene fusions are induced within epithelial cells and macrophages in cell culture. Fusion strains will also be tested for ability to invade, survive and multiply within these cells, as well as for virulence in mice. The proposed research will, therefore, provide important information on Salmonella starvation survival strategies, and the role of nutrient deprivation in virulence of this organism.